Biomass axial distribution in airlift bioreactor with yeast and plant cells

This study was aimed at determining the degree of biomass homogeneity in the various parts of an internal loop airlift bioreactor, thus verifying the assumption, often made in bioreactor studies, of a well-mixed liquid-biomass system. Following characterization of the hydrodynamics of the vessel with water, the axial biomass distribution in the riser and downcomer was determined for plant and yeast cell suspensions of 5.8, 8.5, and 12.5 g DW/L Phaseolus vulgaris and of 30 and 46 g DW/L Saccharomyces cerevisiae. The airlift bioreactor with a surface ratio A(D)/A(D) of 1.04 and aspect ratio of 4.95 was investigated under various aeration rates. The yeast cells were found to be distributed practically uniformly throughout the vessel at the aeration rates of 0.1-1.45 vvm. However, in the case of the denser and cluster-forming plant cells, a clear trend of a gradual bio-mass accumulation in the downcomer, a slightly lower but uniform biomass loading in the riser, and a slightly higher biomass concentration in the gas-liquid separator was observed at the lower aeration rates of 0.1-0.61 vvm. In the case of powderized calcium carbonate (55g/L) often used in fermentations of organic acids, a slight trend of a gradual accumulation of solids towards the bottom parts in both the downcomer and riser was observed. A better representative sampling location, in terms of solids and biomass loading, seems to be in the middle part of the vessel. It is suggested that airlift bioreactors with higher aspect ratios (>5) may be prone to a more significant inhomogeneity of solids (biomass and particles).     

Investigating the action of the microalgal pigment marennine on Vibrio splendidus by in vivo 2H and 31P solid-state NMR

This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo ²H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(?) bacteria membranes. We also investigated the effect of marennine using ³¹P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis.     

In-Cell Solid-State NMR: An Emerging Technique for the Study of Biological Membranes

Biological molecular processes are often studied in model systems, which simplifies their inherent complexity but may cause investigators to lose sight of the effects of the molecular environment. Information obtained in this way must therefore be validated by experiments in the cell. NMR has been used to study biological cells since the early days of its development. The first NMR structural studies of a protein inside a cell (by solution-state NMR) and of a membrane protein (by solid-state NMR) were published in 2001 and 2011, respectively. More recently, dynamic nuclear polarization, which has been used to enhance the signal in solid-state NMR, has also been applied to the study of frozen cells. Much progress has been made in the past 5 years, and in this review we take stock of this new technique, which is particularly appropriate for the study of biological membranes.     

Hydrodynamic vaccination with DNA encoding an immunologically privileged retinal antigen protects from autoimmunity through induction of regulatory T cells

The eye is an immunologically privileged organ whose Ags serve as targets for experimental autoimmune uveitis (EAU), a model for human uveitis. We used a hydrodynamic i.v. injection of naked DNA to express the uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP) in the periphery, thus revoking its immune-privileged status. IRBP was expressed in the liver within hours of administration of as little as 10 microg of IRBP-DNA. Vaccinated mice were highly protected from EAU induced by immunization with IRBP for at least 10 wk after vaccination. Protection was partial in a reversal protocol. Mechanistic studies revealed specific hyporesponsiveness to IRBP without immune deviation, no evidence for apoptosis either by the Fas- or Bcl-2-regulated (mitochondrial) pathway and apparent lack of dependence on CD8(+) cells, IL-10, or TGF-beta. In contrast, depletion of CD25(+) cells after vaccination and before challenge markedly abrogated protection. IRBP-specific CD4(+)CD25(high) T cells could be cultured from vaccinated mice and transferred protection to unvaccinated, EAU-challenged recipients. In vitro characterization of these cells revealed that they are Ag specific, anergic, express FoxP3, CTLA-4, and glucocorticoid-induced TNFR, and suppress by contact. Thus, expression of IRBP in the periphery by DNA vaccination results in tolerance that acts at least in part through induction of IRBP-specific, FoxP3(+)CD4(+)CD25(+) regulatory T cells. DNA vaccination may offer a new approach to Ag-specific therapy of uveitis.     

Comparison of Effects of Compost Amendment and of Single-Strain Inoculation on Root Bacterial Communities of Young Cucumber Seedlings

Compost amendment and inoculations with specific microorganisms are fundamentally different soil treatment methods, commonly used in agriculture for the improvement of plant growth and health. Although distinct, both methods affect the rhizosphere and the plant roots. In the present study we used a 16S rRNA gene approach to achieve an overview of early consequences of these treatments on the assemblage of plant root bacterial communities. For this purpose, cucumber seedlings were grown, under controlled conditions, in perlite potting mix amended with biosolid compost or straw compost, or inoculated with Streptomyces spp. A uniform trend of response of root bacterial communities for all treatments was observed. Root bacterial density, measured as bacterial targets per plant tef gene by real-time PCR, was reduced in 31 to 67%. In addition, increased taxonomic diversity accompanied shifts in composition (α-diversity). The magnitude of change in these parameters relative to the perlite control varied between the different treatments but not in relation to the treatment method (compost amendments versus inoculations). Similarity between the compositions of root and of potting mix bacterial communities (β-diversity) was relatively unchanged. The abundance of Oxalobacteraceae was >50% of the total root bacterial community in the untreated perlite. Root domination by this group subsided >10-fold (straw compost) to >600-fold (Streptomyces sp. strain S1) after treatment. Thus, loss of dominance appears to be the major phenomenon underlining the response trend of the root bacterial communities.Environmental concern over conventional agricultural fertilization and disease control measures has led to increased interest in finding environmentally friendly alternatives. The most explored ones include compost amendments (18, 36) and the application of different microbial preparations (11, 19, 37). These are widely distinct applications. The first approach adds to the amended medium not only a rich and diverse consortium of biological agents but also organic matter and nutrients. It was confirmed that the efficacy of such treatments involves the response of the soil, the plant, and the rhizosphere microbial communities (19, 56). The activities of rhizosphere microorganisms alter the rhizosphere and thus affect plant health and root growth and development (24). Therefore, one of the main objectives of compost amendment or of inoculation with specific microbial strains is manipulation of the plant rhizosphere conditions, particularly via manipulation of the microbial community composition (32).The response of rhizosphere bacterial communities to different anthropogenic and other disturbances has been discussed in terms of resilience (3, 32). Generally, the introduction of new microorganisms produces only restricted spatial and temporal effects on the soil, rhizosphere, and root microbial communities (4, 29, 35). Thus, the plant growth-promoting effect of such treatments may be related to microbial events occurring during the early stages of plant development. Such early effects were pointed out for inoculants of different bacterial species (14, 42) and for compost amendment (15, 21, 50).Consequences of compost amendment or of single species inoculation often include shifts in the plant roots hormonal balance or a plant systemic response, namely, induced systemic resistance (8, 38, 52). Thus, direct or indirect activities of the introduced microorganisms may result in similar modifications of the root habitat. If so, bacterial assemblages of treated roots may share qualitative and quantitative characteristics different from those exhibited by untreated roots.The objective of the present study was therefore to describe and compare responses of bacterial communities of young plant roots to the application of compost or bacterial inoculants. This was performed in a simple model comprised of cucumber seedlings grown in potting mixes amended with compost or inoculated with Streptomyces spp. isolated from the two different composts.     

Stench and sensibilities: On living with waste,animals and microbes in India

Stench is often the most immediate mark of something dirty, decaying and diseased. In India, stench and the smell of acrid smoke commonly indicate the proximity of an open dump or landfill. Frequently a slum is located in the vicinity too, housing waste‐pickers who forage in these sprawling dumps for salvageable waste. These spaces are also host to vermin, birds, stray dogs, pigs, cows and, more recently, dangerous bacteria resistant to even top‐end antibiotics, popularly known as ‘superbugs’. In this paper I examine the socio‐ecological context of neighbourhood, community open garbage dumps and larger landfills in an effort to understand these as part of a dynamic ecosystem of ‘more‐than‐human’ relations. Perceptual variations of smell as experienced in and around waste (in its solid, liquid and gas states) are intrinsically linked to symbolic and material practices across species. Additionally, I suggest that one productive way to think about the emergence of disease and pathogenicity is by considering the information stimulated by smell, which is mediated by cultural interpretations, biological capacities and wider political economies.     

A Comparative Study of the Involvement of 17 Arabidopsis Myosin Family Members on the Motility of Golgi and Other Organelles

Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.The Arabidopsis (Arabidopsis thaliana) myosin gene family contains 17 members: myosin group XI, which includes 13 members (myosins XI-A, -B, -C, -D, -E, -F, -G, -H, -I, -J, and -K, MYA1, and MYA2), and myosin group VIII, which includes four members (ATM1, ATM2, myosin VIIIA, and myosin VIIIB). Both groups are related to unconventional myosin V (Berg et al., 2001; Foth et al., 2006). The Arabidopsis myosins contain a conserved motor domain with ATPase and actin-binding activities, a number of IQ domains that bind myosin light chains, a coiled-coil domain for dimerization, and a specific tail that binds different cargo (Kinkema and Schiefelbein, 1994; Tominaga et al., 2003). Using these functional domains, myosins convert chemical energy from ATP hydrolysis into physical movement along actin fibers, carrying with their tails membrane-bound organelles or RNA/protein complexes (Li and Nebenführ, 2008b).Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (Shimmen and Yokota, 2004; Esseling-Ozdoba et al., 2008), plasmodesmata function (Baluska et al., 2001; Volkmann et al., 2003), organelle movement (Nebenführ et al., 1999; Jedd and Chua, 2002), cytokinesis (Molchan et al., 2002; Collings et al., 2003; Volkmann et al., 2003), endocytosis (Volkmann et al., 2003; Baluska et al., 2004; Samaj et al., 2005), and targeted RNA transport (Hamada et al., 2003). Actomyosin mediated cytoplasmic streaming found in various algae cells reach velocities of up to 100 μm s⁻¹, which is the fastest known myosin-mediated movement (Shimmen and Yokota, 1994).The information that exists regarding specific roles of each plant myosin is rather limited. Immunolocalization studies indicated that myosin XIs are associated with various particles in lily (Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes (Yokota et al., 1995), with mitochondria, plastids, and low-density membranes in maize (Zea mays) root cells (Liu et al., 2001; Wang and Pesacreta, 2004), and with endoplasmic reticulum (ER) in tobacco BY2 cells (Yokota et al., 2008). Specific antibodies against MYA2 showed that it is associated with peroxisomes in epidermal and guard cells of Arabidopsis leaves (Hashimoto et al., 2005). More recent studies using recombinant DNA fusions to fluorescent proteins showed localization of the tails of MYA2, MYA1, XI-K, and XI-I to peroxisomes (Li and Nebenführ, 2007; Reisen and Hanson, 2007) and MYA1 partially localized to Golgi (Li and Nebenführ, 2007). Furthermore, it was shown that peroxisomes, Golgi, and mitochondrial motility were arrested by dominant negative mutants of myosin XI-K and myosin XI-E (Avisar et al., 2008b; Sparkes et al., 2008). Arrest of organelle motility was also found in Arabidopsis knockout plants xi-k and mya2 (Peremyslov et al., 2008) and double mutants xi-k/mya1, xi-k/mya2, and mya2/xi-b (Prokhnevsky et al., 2008). In contrast, the association of the single globular tail domain of MYA1 or MYA2 with peroxisomes did not arrest their motility (Li and Nebenführ, 2007). Knockout plants for myosin xi-k and mya2 had root hair phenotypes (Ojangu et al., 2007; Peremyslov et al., 2008); however, all other 11 myosin XI single knockouts looked normal under regular growth conditions (Peremyslov et al., 2008). Reciprocal stimulation between dimerization via the coiled-coil domains of MYA1 and organelle binding was suggested (Li and Nebenführ, 2008a). As for myosin VIII, immunostaining studies showed that it localized to the cell periphery at plant-specific structures such as plasmodesmata and cytokinetic cell plates (Reichelt et al., 1999; Baluska et al., 2001). Recent data from our laboratory and from others confirmed the presence of myosin VIII in plasmodesmata (Golomb et al., 2008) and the cell plate (Van Damme et al., 2004) and further provided evidence for its involvement with endocytosis (Golomb et al., 2008; Sattarzadeh et al., 2008) and its colocalization with the ER (Golomb et al., 2008). In addition, it was shown that myosin VIII is involved in the plasmodesmata targeting of the beet yellows virus protein Hsp70h (Avisar et al., 2008a).We have determined the role of all 17 genes through transient overexpression of dominant negative forms in leaf epidermal cells. Fluorescent dominant negative fusions not only provide data on the subcellular location but also provide a relatively easy way of determining expression. Additionally, overexpression of dominant negative forms can expose a role of an individual member, which might be masked by redundant activity, if it was silenced. In order to undertake such a large-scale study, we needed to choose an efficient, fast, and reproducible expression system. Therefore, Agrobacterium tumefaciens-mediated transient expression in Nicotiana leaves was suitable.     

The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells

The Bacillus thuringiensis delta-endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species. Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells. The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied. About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae. These results suggest differential interactions of the two toxins with BBMV encompassing all three domains. Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher Bmax and lower Kd than Cry1C domain II and further supported the existence of toxin multisite interactions. Competitive binding assays were used to estimate the sequence of interaction events. Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site. No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins. Taken together, all three domains specifically interact with the epithelial cell membrane. The folding of the three-domain toxin probably dictates the sequence of interaction events.     

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance

 It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band ³¹P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters ≤30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From ³¹P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles. Received: 2 September 1999 / Revised version: 27 February 2000 / Accepted: 27 February 2000     

Stabilization of microbial cytochrome P-450 activity by creation of station-phase conditions in a continuously operated immobilized-cell reactor.

Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-steroid hydroxylation. This activity belongs to the widespread ferredoxin reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers. Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450-based detoxification. It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor. The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in "freezing" of a physiological steady-state analog under stationary phase conditions, allowing stable performance of continuous hydroxylation for several weeks. It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities.